Signal transduction pathways are usually avoided when optimizing a biotransformation process beca... more Signal transduction pathways are usually avoided when optimizing a biotransformation process because they require complex mathematical formulations. The aim of this work was to use a Systems Biology approach to optimize and monitor the biotransformation of l-carnitine using signal transduction pathways. To this end, a dynamic model was constructed, integrating the metabolic pathways of l-carnitine biosynthesis as well as the expression of this metabolism by means of its regulation by transcription factors such as cAMP-CRP and CaiF. The model was validated using different C-sources as well as different reactor feeding approaches. A linear relationship between the external cellular cAMP and the l-carnitine production levels was predicted before being experimentally confirmed in several scenarios. Moreover, results of the model simulations and subsequent experimental findings demonstrated that the addition of exogenous cAMP was able to restore the l-carnitine production when glucose was used as C-source. Additionally, a way to monitor the l-carnitine biosynthesis by using the level of cAMP as a marker of the biotransformation state was in silico and experimentally demonstrated.
The application of metabolic engineering principles to the rational design of microbial productio... more The application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to make quantitative descriptions of the systemic ability of the central carbon metabolism to redirect fluxes to the product-forming pathways. The aim of this work was to further our understanding of the steps controlling the biotransformation of trimethylammonium compounds into L-carnitine by Escherichia coli. Despite the importance of L-carnitine production processes, development of a model of the central carbon metabolism linked to the secondary carnitine metabolism of E. coli has been severely hampered by the lack of stoichiometric information on the metabolic reactions taking place in the carnitine metabolism. Here we present the design and experimental validation of a model which, for the first time, links the carnitine metabolism with the reactions of glycolysis, the tricarboxylic acid cycle and the pentose-phosphate pathway. The results demonstrate a need for a high production rate of ATP to be devoted to the biotransformation process. The results demonstrate that ATP is used up in a futile cycle, since both trimethylammonium compound carriers CaiT and ProU operate simultaneously. To improve the biotransformation process, resting processes as well as CaiT or ProU knock out mutants would yield a more efficient system for producing L-carnitine from crotonobetaine or D-carnitine.
In this work metabolic engineering strategies for maximizing l-(–)-carnitine production by Escher... more In this work metabolic engineering strategies for maximizing l-(–)-carnitine production by Escherichia coli based on the Biochemical System Theory (1–3) and the Indirect Optimization Method are presented (4). The model integrates the metabolic and the bioreactor levels using power-law formalism. Based on the S-system model, the Indirect Optimization Method was applied, leading to profiles of parameter values that are compatible with both the physiology of the cells and the bioreactor system operating conditions. This guarantees their viability and fitness and yields higher rates of l-(–)-carnitine production. Experimental results using a high cell density reactor were compared with optimized predictions from the Indirect Optimization Method. When two parameters (the dilution rate and the initial crotonobetaine concentration) were directly changed in the real experimental system to the prescribed optimum values, the system showed better performance in l-(–)-carnitine production (74% increase in production rate), in close agreement with the modelapos;s predictions. The model shows control points at macroscopic (reactor operation) and microscopic (molecular) levels where conversion and productivity can be increased. In accordance with the optimized solution, the next logical step to improve the l-(–)-carnitine production rate will involve metabolic engineering of the E. coli strain by overexpressing the carnitine transferase, CaiB, activity and the protein carrier, CaiT, responsible for substrate and product transport in and out of the cell. By this means it is predicted production may be enhanced by up to three times the original value.
Signal transduction pathways are usually avoided when optimizing a biotransformation process beca... more Signal transduction pathways are usually avoided when optimizing a biotransformation process because they require complex mathematical formulations. The aim of this work was to use a Systems Biology approach to optimize and monitor the biotransformation of l-carnitine using signal transduction pathways. To this end, a dynamic model was constructed, integrating the metabolic pathways of l-carnitine biosynthesis as well as the expression of this metabolism by means of its regulation by transcription factors such as cAMP-CRP and CaiF. The model was validated using different C-sources as well as different reactor feeding approaches. A linear relationship between the external cellular cAMP and the l-carnitine production levels was predicted before being experimentally confirmed in several scenarios. Moreover, results of the model simulations and subsequent experimental findings demonstrated that the addition of exogenous cAMP was able to restore the l-carnitine production when glucose was used as C-source. Additionally, a way to monitor the l-carnitine biosynthesis by using the level of cAMP as a marker of the biotransformation state was in silico and experimentally demonstrated.
The application of metabolic engineering principles to the rational design of microbial productio... more The application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to make quantitative descriptions of the systemic ability of the central carbon metabolism to redirect fluxes to the product-forming pathways. The aim of this work was to further our understanding of the steps controlling the biotransformation of trimethylammonium compounds into L-carnitine by Escherichia coli. Despite the importance of L-carnitine production processes, development of a model of the central carbon metabolism linked to the secondary carnitine metabolism of E. coli has been severely hampered by the lack of stoichiometric information on the metabolic reactions taking place in the carnitine metabolism. Here we present the design and experimental validation of a model which, for the first time, links the carnitine metabolism with the reactions of glycolysis, the tricarboxylic acid cycle and the pentose-phosphate pathway. The results demonstrate a need for a high production rate of ATP to be devoted to the biotransformation process. The results demonstrate that ATP is used up in a futile cycle, since both trimethylammonium compound carriers CaiT and ProU operate simultaneously. To improve the biotransformation process, resting processes as well as CaiT or ProU knock out mutants would yield a more efficient system for producing L-carnitine from crotonobetaine or D-carnitine.
In this work metabolic engineering strategies for maximizing l-(–)-carnitine production by Escher... more In this work metabolic engineering strategies for maximizing l-(–)-carnitine production by Escherichia coli based on the Biochemical System Theory (1–3) and the Indirect Optimization Method are presented (4). The model integrates the metabolic and the bioreactor levels using power-law formalism. Based on the S-system model, the Indirect Optimization Method was applied, leading to profiles of parameter values that are compatible with both the physiology of the cells and the bioreactor system operating conditions. This guarantees their viability and fitness and yields higher rates of l-(–)-carnitine production. Experimental results using a high cell density reactor were compared with optimized predictions from the Indirect Optimization Method. When two parameters (the dilution rate and the initial crotonobetaine concentration) were directly changed in the real experimental system to the prescribed optimum values, the system showed better performance in l-(–)-carnitine production (74% increase in production rate), in close agreement with the modelapos;s predictions. The model shows control points at macroscopic (reactor operation) and microscopic (molecular) levels where conversion and productivity can be increased. In accordance with the optimized solution, the next logical step to improve the l-(–)-carnitine production rate will involve metabolic engineering of the E. coli strain by overexpressing the carnitine transferase, CaiB, activity and the protein carrier, CaiT, responsible for substrate and product transport in and out of the cell. By this means it is predicted production may be enhanced by up to three times the original value.
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Papers by Angel Sevilla